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Respiratory Research
Open Access
Research
Hormonal regulation of alveolarization: structure-function
correlation
Samuel J Garber
†1
, Huayan Zhang
†1
, Joseph P Foley
1
, Hengjiang Zhao
1
,
Stephan J Butler
1
, Rodolfo I Godinez
2
, Marye H Godinez
2
, Andrew J Gow
1

and Rashmin C Savani*
3
Address:
1
Division of Neonatology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine,

Philadelphia, PA, USA,
2
Department of Anesthesiology and Critical Care Medicine, Children's Hospital of Philadelphia, University of
Pennsylvania School of Medicine, Philadelphia, PA, USA and
3
Division of Neonatal-Perinatal Medicine, Division of Pulmonary and Vascular
Biology, Room K4.224, University of Texas Southwestern at Dallas, Dallas, TX USA
Email: Samuel J Garber - ; Huayan Zhang - ; Joseph P Foley - ;
Hengjiang Zhao - ; Stephan J Butler - ; Rodolfo I Godinez - ;
; Andrew J Gow - ;
Rashmin C Savani* -
* Corresponding author †Equal contributors
Abstract
Background: Dexamethasone (Dex) limits and all-trans-retinoic acid (RA) promotes alveolarization. While structural changes
resulting from such hormonal exposures are known, their functional consequences are unclear.
Methods: Neonatal rats were treated with Dex and/or RA during the first two weeks of life or were given RA after previous
exposure to Dex. Morphology was assessed by light microscopy and radial alveolar counts. Function was evaluated by
plethysmography at d13, pressure volume curves at d30, and exercise swim testing and arterial blood gases at both d15 and d30.
Results: Dex-treated animals had simplified lung architecture without secondary septation. Animals given RA alone had smaller,
more numerous alveoli. Concomitant treatment with Dex + RA prevented the Dex-induced changes in septation. While the
results of exposure to Dex + RA were sustained, the effects of RA alone were reversed two weeks after treatment was stopped.
At d13, Dex-treated animals had increased lung volume, respiratory rate, tidal volume, and minute ventilation. On d15, both
RA- and Dex-treated animals had hypercarbia and low arterial pH. By d30, the RA-treated animals resolved this respiratory
acidosis, but Dex-treated animals continued to demonstrate blood gas and lung volume abnormalities. Concomitant RA
treatment improved respiratory acidosis, but failed to normalize Dex-induced changes in pulmonary function and lung volumes.
No differences in exercise tolerance were noted at either d15 or d30. RA treatment after the period of alveolarization also
corrected the effects of earlier Dex exposure, but the structural changes due to RA alone were again lost two weeks after
treatment.
Conclusion: We conclude that both RA- and corticosteroid-treatments are associated with respiratory acidosis at d15. While
RA alone-induced changes in structure andrespiratory function are reversed, Dex-treated animals continue to demonstrate

increased respiratory rate, minute ventilation, tidal and total lung volumes at d30. Concomitant treatment with Dex + RA
prevents decreased septation induced by Dex alone and results in correction of hypercarbia. However, these animals continue
to have abnormal pulmonary function and lung volumes. Increased septation as a result of RA treatment alone is reversed upon
discontinuation of treatment. These data suggest that Dex + RA treatment results in improved gas exchange likely secondary
to normalized septation.
Published: 27 March 2006
Respiratory Research2006, 7:47 doi:10.1186/1465-9921-7-47
Received: 27 May 2005
Accepted: 27 March 2006
This article is available from: />© 2006Garber et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( />),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Respiratory Research 2006, 7:47 />Page 2 of 12
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Background
Bronchopulmonary Dysplasia (BPD) remains a signifi-
cant cause of morbidity and mortality affecting as many as
30–40% of infants born less than 30 weeks gestation [1].
While the pathophysiology of BPD includes both inflam-
matory and fibrotic processes, a critical component is an
arrest of lung development at the saccular stage and fail-
ure of alveolarization [1]. Alveolar hypoplasia has been
documented in preterm humans [2] as well as in preterm
baboons born at 75% gestation and ventilated for the first
two to three weeks of life [3].
Lung development is a dynamic process consisting of
embryonic, pseudoglandular, canalicular, saccular, and
alveolar stages marked by the progression from a rudi-
mentary lung bud to a saccule with a completely devel-
oped respiratory tree. In the human, alveolarization

begins during the 36
th
week of gestation and continues for
at least 3 years after birth [4]. The development of alveoli
involves the formation of crests, or secondary septae, at
precise sites of the saccular wall. These crests protrude into
the saccular air space, include the inner layer of the capil-
lary bilayer, and further subdivide the saccule into subsac-
cules that later become mature alveoli. While not fully
understood, the regulation of this process involves several
cell types including endothelial cells, myofibroblasts, and
epithelial cells as well as growth factors, hormones, and
environmental conditions that either inhibit or stimulate
alveolar growth [5].
The stages of lung development are the same in rodents
except that alveolar formation is an entirely postnatal
event occurring in the first three weeks of life [6,7]. Inter-
estingly, in rodents, alveolarization is associated with
decreased plasma corticosteroid concentrations [8], and
administration of corticosteroids during this period
inhibits alveolarization [9]. Using a neonatal rat model,
Massaro and others have demonstrated the effects of Dex-
amethasone (Dex) and all-trans-retinoic acid (RA) treat-
ment on alveolar development [10]. Dex-treated animals
develop a simplified architecture with large terminal sacs,
whereas RA-treated animals develop smaller, more
numerous alveoli. Dex-induced changes are prevented in
animals that receive either concomitant Dex + RA admin-
istration [10] or RA after earlier treatment with Dex alone,
even though RA is given after the period known to be crit-

ical for alveolar development [11].
While considerable information is available for hormo-
nally mediated structural changes during alveolarization,
there is a paucity of information on the impact of such
hormonal manipulations and the resultant architectural
alterations on pulmonary function. Srinivasan et al. [12]
measured pulmonary function in rats treated with Dex
and/or RA in the first two weeks of life. In their studies,
changes in lung volume and compliance resulting from
Dex treatment alone were not reversed with simultaneous
RA administration [12]. However, since Srinivasan's study
was done in sedated 30–39 day old animals, it is unclear
if functional effects of altered alveolarization are evident
in normally breathing rats. In addition, no information
on arterial oxygen or carbon dioxide homeostasis or exer-
cise tolerance was currently available for this model.
The goal of the current study was to determine the struc-
ture-function relationships after glucocorticoid and retin-
oid treatment in neonatal rat pups undergoing
alveolarization. We report that both RA and Dex-induced
alteration of alveolarization was associated with hypercar-
bia at two weeks. However, only Dex-treated animals had
larger lung volumes with increased respiratory rate and
tidal volume. Concomitant RA treatment prevented the
Dex-induced changes in secondary septation and cor-
rected the respiratory acidosis. However, Dex + RA-treated
animals continued to have increased respiratory rate, tidal
volume, minute ventilation, and larger lung volumes.
Treatment with RA alone increased the number of alveoli
as measured by radial alveolar counts, but this response

was reversed two weeks after stopping treatment, even if
the RA treatment was given later, after the optimal time
for alveolarization.
Methods
Animals
All protocols were reviewed and approved by the Chil-
dren's Hospital of Philadelphia (CHOP) Institutional
Animal Care and Use Committee in accordance with NIH
guidelines. Timed pregnant Sprague-Dawley rats (Charles
River Breeding Laboratory, Wilmington, MA), were main-
tained until parturition on a 12:12 h light:dark cycle with
unlimited access to food (Purina Lab Diet, St. Louis, MO)
and water in the Laboratory Animal Facility at CHOP.
Day 15/30 protocol
After birth, litters were adjusted to 10 pups per litter
within 12 h of birth and divided into the following treat-
ment groups: (1) Dexamethasone (Dex, American Regent
Laboratories, Inc., Shirley, NY) 0.1 µg in 20 µl 0.9%NaCI
[saline]) or saline alone (20 µl) subcutaneously (SQ)
daily from days 1–14; (2) all-trans-retinoic acid (RA,
Sigma-Aldrich, St. Louis, MO) 500 µg/kg in 20 µl cotton-
seed oil (CSO, Sigma-Aldrich, St. Louis, MO) or CSO
alone (20 µl) via intraperitoneal (IP) injection daily days
3–14; (3) Dex and RA at doses and days as above; (4)
saline and CSO at doses and days above; and (5) control
(no injections). The dose of Dex was based on previous
literature demonstrating only mild effects on somatic
growth [9]. Because it was difficult to discern the gender
of rats at birth, both males and females were studied at
days 1,5, 10, 15, and 30 as described below.

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Extended (Day 37/52) study protocol
This study was designed to evaluate the structural conse-
quences of RA administered after the critical period for
alveolar development in rats previously treated with Dex
from days 1 to 14. Pups were normalized to 10 per litter
shortly after birth and, in addition to a control group (no
injections), were divided into the following groups receiv-
ing either saline or Dex SQ daily on days 1–14 followed
by either IP CSO or RA daily on days 24–36: (1) Early
Saline + Later CSO; (2) Early Saline + Later RA; (3) Early
Dex + Later CSO; (4) Later Dex + Later RA. Animals were
weaned from their mothers at d21 and divided by sex into
separate cages (n = 2–3 per cage). The doses of saline, Dex,
and CSO were as above. Males and females were studied
independently as described below on d37 (after 2 weeks
of RA treatment) and d52 (2 weeks after stopping RA treat-
ment).
Lung harvest
Anesthesia for all studies was attained using an intramus-
cular injection of a Ketamine/Xylazine (87:13 µg/kg)
cocktail. The right lung was removed, snap frozen in liq-
uid nitrogen, and stored at -80°C for future analysis. As
has been previously described [13], the left lung was
inflated to 25 cm H
2
O pressure with formalin and stored
in formalin for 24 hours. Lungs were then processed to
obtain 5-micron thick paraffin sections.

Structural analyses
Histology
For each time point, sections were stained with hematox-
ylin and eosin in order to examine lung architectural dif-
ferences using light microscopy. Both 40x and 100x
images were obtained using a Nikon TE 300 inverted
microscope.
Radial alveolar counts (RAC)
To quantify alveolarization, RAC were obtained as
described by Emery and Mithal [14] and validated by
Cooney and Thurlbeck [15]. These investigators con-
firmed that forty measurements per lung were sufficient to
establish a reliable morphometric assessment of alveolari-
zation. Briefly, a perpendicular line was drawn from the
last respiratory bronchiole to either the pleura or the near-
est connective tissue septum. Using low power images,
over 90% of all lines drawn were to the pleura. A mini-
mum of forty lines for each lung were drawn and the
number of septae intersected were counted for each line.
In addition, at least three sections from several levels
within the tissue block were used for each animal.
Functional analyses
Plethysmography
On d13, pups were placed in a dual chamber plethysmo-
graph (Buxco Electronics Inc, Sharon, CT) for non-inva-
sive, non-sedated, real-time measurement of pulmonary
function. This airtight system, which separates the head
from the body by a latex collar barrier, measures airflow
across a pneumotach plate and uses a flow transducer to
determine various parameters including respiratory rate

(RR), tidal volume (TV), and minute ventilation (MV).
Animals were acclimated to the chamber until consist-
ently normal breathing patterns were noted. Thermal neu-
trality was maintained throughout the study period for
each animal. Measurements were made twice, each for
two minutes with only data that were consistently within
5% variance of each other used for analysis. TV and MV
were normalized to body weight. We were unable to
obtain measurements at d30 as the rats were too large for
the dual chamber plethysmograph.
Arterial blood gases (ABG)
To evaluate the efficiency of gas exchange, an ABG was
obtained from the distal aorta at the time of harvest for
d15 and d30 animals. While animals were spontaneously
breathing under adequate anesthesia, the abdomen was
opened. With the diaphragm left intact, the distal aorta
was identified, and a sample drawn using a heparinized
syringe. The harvest then proceeded as described above.
Samples were analyzed using an i-STAT Portable Clinical
Analyzer (i-STAT Corporation, East Windsor, NJ).
Lung volume of displacement
At d15, lungs were inflated to a pressure of 25 cm H2O
with 10% formalin, harvested en bloc and fixed over-
night. Lung volume was measured by waterdisplacement
immediately after inflation with maintenance of inflation
confirmed by repeat measurement 24 hours after fixation.
Pressure-Volume (PV) studies
Separate animals were studied at d30 to obtain PV curves.
After appropriate anesthesia, the trachea was cannulated
and the animals were placed on a Harvard rodent ventila-

tor (Harvard Apparatus Inc., Holliston, MA). Animals
were ventilated with 100% O
2
for 10 minutes after which
time the cannula was sealed by closing the stopcock to
allow the lungs to degas. PV curves were obtained with the
chest closed. Inflation and deflation of the lungs was per-
formed in 0.5 ml air increments and pressure was meas-
ured by an HP Omni Care (Wolfpham, MA) using an
Abbott pressure transducer (HP M1006B pressure modu-
lator, North Chicago, IL). Maximum inflation was
achieved at 33 mmHg (25 cm H
2
O) and maximum defla-
tion was achieved by the corresponding withdrawal of air
to decrease pressure to 0 mmHg. Only lungs that did not
leak were included for analysis.
Analysis of PV curves
Regression analysis using Sigma Plot 8.0 (Systat Software
Inc., Port Richmond, CA) generated best-fit models for
Respiratory Research 2006, 7:47 />Page 4 of 12
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inflation and deflation curves using data for all animals in
each treatment group. For inflation, a sigmoidal 3 param-
eter model was utilized [ ] where
"y" is the lung volume, "a" is an estimate of the maximum
lung volume (Vmax), "x" is the pressure at a given vol-
ume, "x
0
" is the pressure at a volume of 0, and "b" is a con-

stant. The deflation model was based on an exponential
rise model [y = a(1-e
-bx
)]. The parameters within this
model provided an estimate of Vmax and the standard
error of the estimate. First derivative curves were used to
determine maximum compliance and the pressure at
which this was achieved, while second derivative curves
were used to calculate points of maximum acceleration
and deceleration during inflation and deflation. Hystere-
sis was defined as the area bound by the inflation and
deflation curves. To quantify differences between treat-
ment groups, the area was obtained and averaged for each
treatment group. All parameters were adjusted to body
weight in kilograms.
Forced exercise swim testing
Separate groups of animals underwent forced swim test-
ing to evaluate exercise tolerance at d15 and d30. Rats
were placed in a tank filled with water at 24°C at a level
high enough to prevent their tails from touching the bot-
tom of the tank. They remained in the water until the first
sign of fatigue manifested by their entire body sinking
below the water level. They were then rescued and har-
vested a day later as described above.
Statistical analysis
For all variables measured, values were expressed as mean
± SE, using the number of animals rather than the number
of observations for calculations. ANOVA and two-tailed t-
tests assuming unequal variances with Tukey correction
were used to determine intergroup significance with a p-

value <0.05 considered statistically significant for all anal-
yses.
To analyze PV curves, z-scores were used for comparison
of Vmax between treatment groups with a score >1.96
considered significant at p < 0.05. For analysis of maxi-
mum compliance and pressure at which it was achieved,
rate of maximum deflation, and hysteresis, an ANOVA
and unpaired t-test with Tukey correction were used with
p < 0.05 considered significant for all analyses.
Results
Day 15/30 protocol
Neonatal rats were exposed to either saline or Dex and/or
CSO or RA for the first two weeks of life as described in
Methods. We first sought to reproduce the structural alter-
ations from hormonal treatments during the critical
period of alveolar development in rats [10]. Animals were
examined at postnatal days 1, 5, 10, 15, and 30.
Weight gain
All animals were the same weight at the start of the exper-
iment (6.7 ± 0.1 grams, n = 60), and litter sizes were nor-
malized to 10 per litter to ensure equal access to nutrition.
Table 1 shows the weights of rats given various treatments
throughout the first two weeks and at d30 of life. Dex- and
Dex + RA-treated pups had significantly less weight gain
compared to saline- or Saline + CSO-treated animals by
day 5 (Table 1). At d 15, Dex and Dex + RA pups weighed
approximately 15% less than corresponding controls. Rats
treated with RA alone had weight gain comparable to con-
trol animals at all time points. Despite stopping hormo-
nal treatments at d14, the body weights of Dex- and Dex

+ RA-treated animals continued to be significantly lower
(about 20%) than controls at d30 (Table 1).
Morphology
Hormonal treatment of rat pups during the period of alve-
olar development resulted in alterations of lung architec-
ture. At d15, Dex-treated animals appeared to have larger,
simpler distal air spaces than saline controls. These struc-
tural changes were evident as early as d5 (Figures 1 and 2)
and, despite discontinuation of treatment at d14, per-
sisted to d30. RA-treated pups, on the other hand,
appeared to have smaller, more numerous alveoli than
ya e
xx b
=+
−−
/( )
(( )/ )
1
0
Table 1: Body weights in grams: Though no differences in body weight were noted at birth between groups (6.7 ± 0.1 grams, n = 60),
the effect of Dex on weight gain was evident by day 5 and continued until d30 as both Dex and Dex + RA pups had significantly lower
weights compared to saline controls. RA treatment alone did not affect weight. Values are expressed mean ± SE. *p < 0.05 vs.
corresponding controls.
Treatment Group Day 5 Day 10 Day 15 Day 30
Saline/Saline + CSO (n = 8–15) 12.9 ± 0.4 17.1 ± 0.4 31.4 ± 0.8 164 ± 7
Cottonseed oil (CSO) (n = 8–15) 13.2 ± 0.3 16.6 ± 0.6 29.1 ± 1.0 139 ± 13
Retinoic Acid (RA) (n = 8–15) 12.9 ± 0.3 16.8 ± 0.4 28.8 ± 0.8 150 ± 8
Dexamethasone (Dex) (n = 8–16) 11.4 ± 0.2* 15.0 ± 0.2* 25.8 ± 0.7* 126 ± 6*
Dex + RA(n = 7–14) 11.7 ± 0.2* 15.4 ± 0.3* 26.0 ± 0.9* 125 ± 10*
Respiratory Research 2006, 7:47 />Page 5 of 12

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CSO controls as early as d5 and up to d15. Interestingly,
rats treated with RA alone up to 14 days and examined at
d30 had lung histology similar to that of control animals
(Figure 2), demonstrating a loss of the RA effect within
two weeks of stopping treatment. Meanwhile, Dex + RA-
exposed pups showed a simplified distal architecture sim-
ilar to Dex alone pups at days 5 and 10. The corticoster-
oid-induced changes in architecture were prevented by
days 10 to 15 with concomitant RA treatment such that, at
d15, they displayed a distal lung structure similar to that
of controls. In contrast to animals treated with RA alone,
the effect of concomitant Dex + RA treatment was sus-
tained to d30 (Figure 2).
Radial alveolar counts (RAC)
Morphometric evaluation of alveolarization was achieved
using RAC. (Figure 3). Compared to controls, and in
accordance with histological appearance, RAC were signif-
icantly lower in Dex-treated and significantly higher in
RA-treated pups at days 5, 10, and 15. Dex + RA animals
had lower RAC compared to saline controls at days 5 and
10, but by day 15, rats treated with both hormones had
RAC that were similar to controls (Figure 3). At d30, while
RAC remained significantly lower in Dex-treated animals
compared to saline controls, the RAC for both RA- and
Changes in morphology during hormonal treatments at days 1, 5, 10, 15, and 30: Dex-induced changes in architecture were evident as early as d5 and persisted to d30Figure 1
Changes in morphology during hormonal treatments at days 1, 5, 10, 15, and 30: Dex-induced changes in architecture were
evident as early as d5 and persisted to d30. RA-induced changes were also evident at d5, continued to d15, but had reversed at
d30. Concomitant Dex and RA administration resulted in septation similar to that of controls between d10 and d15 with con-
tinued normal appearance at d30. Dex: Dexamethasone. RA: all-trans-retinoic acid, (all images 40× magnification)

Table 2: Radial alveolar counts (RAC) at d15 and d30: RAC at d15
were significantly lower in Dex-treated and higher in RA-treated
pups while Dex + RA animals were similar to controls. At d30,
RAC continued to be significantly lower in Dex-treated pups but
RA alone increases were lost demonstrating reversal upon
stopping treatment. Values are expressed mean ± SE. *p < 0.05
vs. saline-treated animals.
†
p < 0.01 vs. CSO-treated animals.
‡
p <
0.01 vs. d15 RA animals.
Treatment Group Day 15 Day 30
Control (n = 3–4) 8.5 ± 0.2 9.0 ± 0.3
Saline (n = 3–4) 8.7 ± 0.3 8.8 ± 0.3
Cottonseed oil (CSO) (n = 3–4) 8.5 ± 0.2 8.8 ± 0.1
Retinoic Acid (RA) (n = 3–4) 11.6 ± 0.2
†
8.9 ± 0.1
‡
Dexamethasone (Dex) (n = 3–4) 6.6 ± 0.2* 7.3 ± 0.4*
Dex + RA (n = 3–4) 8.9 ± 0.3 8.5 ± 0.2
Respiratory Research 2006, 7:47 />Page 6 of 12
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Dex + RA-treated animals were similar to controls (Table
2).
Plethysmography
In order to determine the functional consequences of hor-
monally altered lung architecture, a number of variables
of pulmonary function were examined at d13 (Table 3).

In association with decreased RAC, Dex-treated animals
had a significantly increased RR, TV, and MV compared to
saline controls. While concomitant treatment with RA
(Dex + RA) reversed RAC as compared to Dex alone, this
treatment had no effect on the increased RR, MV, or TV
seen in association with Dex alone. RA alone, a treatment
that increased RAC, had no effect on RR, MV, or TV (Table
3).
ABG
Since significantly increased RR, MV, and TV were
observed on d13 in Dex- and Dex + RA-treated animals,
we examined ABGs to evaluate gas exchange both at d15
and d30 (Table 4). On d15, when RA alone and Dex alone
treatment altered distal lung architecture, both sets of rat
pups had hypercarbia with respiratory acidosis. On d30,
when the RA alone-treated animals had RAC similar to
those of controls, the RA-alone animals had normal pH
and PCO
2
. Also at d30, Dex alone-treated animals, that
had persistentlylarger distal air spaces, continued to have
hypercarbia with a respiratory acidosis. However, Dex +
RA-treated animals at both d15 and d30 had pCO
2
values
no different from control despite continued increased RR,
MV, and TV. Interestingly, oxygenation was lower in the
d15 group given RA alone. Dex + RA animals at d15, how-
ever, had PO
2

values no different from those of controls
(Table 4). These data suggest impaired gas exchange in
Dex alone-treated animals with a failure of secondary sep-
tation, and, despite continued tachypnea and increased
minute ventilation, a correction of this abnormality
occurred with concomitant RA treatment.
Lung volume of displacement
As hypercarbia could result from increased dead space
with larger lung volumes, we determined the lung vol-
umes of displacement of hormonally treated animals at
d15 of life (Table 5). Both Dex- and Dex + RA-treated pups
had volumes of displacement that were significantly
greater than those of control, saline or RA-treated animals,
suggesting that Dex treatment is associated with larger
lung volumes and that concomitant RA-treatment does
not prevent this.
PV curves
In order to confirm the lung volume of displacement
measurements made at d15 and to evaluate lung volumes
using an independent method, PV curves were generated
on d30 as described in Methods. As shown in Figure 4A
and 4C, Vmax was significantly increased in both Dex and
Dex + RA compared to control/saline (z = 2.05). No dif-
ference existed between Dex compared to Dex + RA curves
(z = 0.13) and Control/Saline versus RA curves (z = 0.6).
The deflation curve for each treatment group was based on
an exponential rise to maximum model (Figure 4B). The
best-fit inflation and deflation curves generated for each
treatment group are shown in Figure 4C1-4, with dots rep-
resenting individual measurements for each animal. A sig-

nificantly increased hysteresis was noted in Dex versus all
other groups (Dex: 739 ± 19; Control/Saline: 566 ± 41;
RA: 572 ± 66; Dex + RA: 594 ± 24 (ml/kg)
2
, p < 0.05, n =
3–6 per group). Collectively, these data suggest that Dex
treatment resulted in larger lung volumes that concomi-
tant RA treatment failed to abrogate.
The pressure required to reach the point of maximum
compliance during inflation was lower in Dex vs. Control/
Saline (16.3 ± 0.3 vs. 18.4 ± 0.6 mm H
2
O/kg, p = 0.014, n
= 3–6 per group) and between RA and Dex + RA curves
(17.9 ± 0.3 vs. 15.3 ± 0.3 mm H
2
O/kg, p < 0.01, n = 3–6
per group). In addition, the rate of maximal deflation was
significantly greater in Dex vs. Control/Saline curves (9.6
± 0.3 vs. 5.5 ± 0.4 mm H
2
O/kg/s, p < 0.05, n = 3–6 per
group). The rate of maximal deflation tended to be lower
in Dex vs. Dex + RA curves but this did not reach signifi-
cance (9.6 ± 0.3 vs. 7.2 ± 0.9 mm H
2
O/kg/s, p = 0.07, n =
3–6 per group). This parameter was similar between RA
and Dex + RA curves (7.4 ± 1.3 vs. 7.2 ± 0.9 mm H
2

O/kg/
s, p = 0.88, n = 3–6 per group). These data suggest that Dex
treatment resulted in lungs that had larger residual vol-
umes requiring higher pressures to achieve the point of
maximal compliance but were less stable during deflation.
Taken together, these physiologic data demonstrate
that Dex + RA treatment failed to prevent larger lung
volumes, RR, MV, and TV seen with Dex treatment
alone. However, CO
2
elimination improved, sug-
gesting better gas exchange with increased septation.
Exercise swim testing
No difference in time to fatigue was noted on forced exer-
cise swim testing for any group of rats (Saline/Control 45
± 2, CSO/RA 45 ± 3, Dex, 45 ± 2, Dex + RA 39 ± 2 minutes,
n = 6–8 per group). This suggests that, even in Dex-treated
animals that demonstrated compromised pulmonary
function by other measures, exercise tolerance was not
affected by hormonal treatments.
Extended study
Since Massaro and Massaro have previously demonstrated
that RA promotes septation after the period of normal
alveolarization [11] and our data showed that early RA
effects were lost two weeks after stopping treatment, we
next sought to determine whether the effects of later
Respiratory Research 2006, 7:47 />Page 7 of 12
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administration of RA were also reversed. Rat pups were
normalized to 10 pups per litter and treated with either

Dex or saline from days 1–14 followed by either CSO or
RA from days 24–36 (12 days of treatment). Animals were
studied at either day 37 (at the end of treatments) or day
52 (2 weeks after stopping treatment).
Weight gain
Birth weights were the same for all animals (6.9 ± 0.1, n =
40). In untreated animals, growth velocities were similar
until d24 after which males grew faster than females such
that by d36 females weighed approximately 10% less than
males (data not shown). Dex treatment affected both
males and females equally with 8–9% lower weight at d14
(p < 0.01, n = 8 per group) and a 6–8% lower weight at
d36 as compared to sex-matched controls (p = 0.09, n = 8
per group). As with the earlier study, RA treatment alone
had no effect on weight (data not shown).
Morphology
Alterations of lung architecture were similar to those seen
with the Day 15/30 protocol (Fig. 5). At d37, Early Dex +
Later CSO-treated animals had simplified distal air spaces
compared to Early Saline + Later CSO controls. Early
Saline + Later RA-treated pups, on the other hand, had
smaller, more numerous alveoli than Early Saline + Later
CSO controls. Normal architecture was restored in Early
Dex + Later RA-exposed rats (Figure 5). Changes seen in
the Early Dex + Later CSO group persisted at d52 while
animals exposed to Early Dex + Later RA continued to
have architecture similar to that of controls. Interestingly,
at d52, Early Saline + Later RA-treated rats had lung histol-
Days 15 (top) and 30 (bottom) histology: A simplified distal architecture was seen in Dex-treated animals at both daysFigure 2
Days 15 (top) and 30 (bottom) histology: A simplified distal architecture was seen in Dex-treated animals at both days. At d15,

RA-treated pups had smaller more numerous alveoli, but these changes were no longer seen at d30. Dex + RA treatment
resulted in a restitution of septation to near that of saline controls at both days, (all images 100× magnification)
Table 3: Plethysmography at d13: Dex-treated animals showed an increased respiratory rate (RR), tidal volume, and minute
ventilation compared to saline controls. Retinoic acid treatment alone did not alter RR but, when given with Dex, resulted in
decreased RR similar to that of controls. Values are expressed mean ± SE. *p < 0.05 vs. saline;
†
p = 0.3 vs. Dex;
‡
p = 0.5 vs. Dex;
§
p = 0.9
vs. Dex. bpm: breaths per minute
Treatment Group Respiratory Rate (bpm) Tidal Volume (ml/kg) Minute Ventilation (ml/kg)
Control (n = 8) 171 ± 20 5.2 ± 0.9 861 ± 145
Saline (n = 9) 180 ± 5.0 4.8 ± 0.8 890 ± 163
Cottonseed oil (CSO) (n = 14) 179 ± 12 6.5 ± 0.7 935 ± 109
Retinoic acid (RA) (n = 15) 180 ± 6.0 5.2 ± 0.7 810 ± 103
Dexamethasone (Dex) (n = 16) 211 ± 11* 8.1 ± 0.8* 2260 ± 150*
Dex + RA (n = 14) 195 ± 9.0
†
8.7 ± 1.0
‡
2131 ± 186
§
Respiratory Research 2006, 7:47 />Page 8 of 12
(page number not for citation purposes)
ogy that appeared similar to that of control animals (Fig-
ure 5), again demonstrating a loss of the effects of RA
alone two weeks after treatment was stopped.
Radial alveolar counts

RAC were used to quantify the changes in alveolarization
in the extended study. In concordance with histological
appearance, RAC were significantly lower in Early Dex +
Later CSO-treated and significantly higher in Early Saline
+ Later RA-treated animals at d37 (Table 6). Rats treated
with both hormones had RAC no different from controls.
At d52, RAC remained significantly lower in Early Dex +
Later CSO-treated animals compared to Early Saline +
Later CSO controls, but both Early Saline + Later RA- and
Early Dex + Later RA-treated animals were similar to con-
trols thereby confirming the reversal of RA alone effects
two weeks after stopping treatment (Table 6). The distri-
bution of males and females in these studies was equal
and no differences were noted between them with respect
to the histology or RAC (data not shown).
Discussion
In the present study, we confirm hormonally mediated
changes in architecture during postnatal lung develop-
ment in the rat. Respiratory acidosis, the most significant
functional abnormality, was noted on d15 in both RA
alone-and Dex alone-treated rat pups and was resolved in
Dex + RA-treated animals. However, Dex + RA failed to
resolve the increased tachypnea, MV, and TV seen in Dex
alone-treated rats. Massaro and Massaro have previously
shown that Dex + RA treatment results in an increased
body mass-specific surface area available for gas exchange
compared to rats treated with Dex alone [10]. In the face
of persistently larger lung volumes and equivalent body
weight in both Dex-and Dex + RA-treated animals, the
improved CO

2
elimination in Dex + RA-treated animals is
likely the effect of improved secondary septation and a
larger surface area for gas exchange.
Interestingly, the increase in RAC on d15 in rats treated
with RA alone was associated with hypercarbia, lower
PaO
2
and acidosis, but without any effect on other pulmo-
nary function parameters studied. The reason for this
abnormality in ABG is unclear, but suggests a defect in gas
exchange. It is unlikely that this abnormality is due to the
increased number of alveoli as it has previously been
shown that RA treatment alone does not increase surface
area [10]. However, ABG were normal in Dex + RA ani-
mals at d15, as well as in RA alone-treated pups by d30
when the RA alone-stimulated changes in distal lung
structure had also resolved. Indeed, while the effects of
Dex alone and concomitant RA administration were sus-
tained for at least 15 days after stopping the treatments,
the effects of RA alone from either d4 to d14 or d24 to d36
were reversed two weeks after stopping RA.
Alveolar development, the last phase of lung develop-
ment, occurs either pre- or postnatally depending on the
species. In the human, alveolarization begins in utero at
about 36 weeks of gestation and continues postnatally,
whereas in the rodent, secondary septation is an entirely
postnatal event. Alveolarization appears to correlate
inversely with changes in serum corticosteroid concentra-
tions. It is likely that the normal timing of alveolar devel-

opment reflects decreased corticosteroid levels leading to
an increase in DNA synthesis and septation. For example,
in the rat, corticosterone concentrations drop to a nadir
between postnatal days 6 and 12, the time of maximum
alveolar formation [8]. Conversely, administration of cor-
ticosteroid during this critical period results in an inhibi-
tion of alveolarization [9]. Indeed, exposure of fetal
rhesus macaques to triamcinolone during the pseudog-
landular and saccular phases of lung development results
in an inhibition of septation [16]. The mechanisms of
Dex-induced inhibition of alveolarization are likely mul-
tifactorial, including inhibition of DNA synthesis, differ-
ential regulation of matrix components, and changes in
gene expression in the lung [17]. In addition, corticoster-
oids cause a growth retardation that is in itself associated
with a slowing of alveolar growth [18].
Radial alveolar counts (RAC) as a percentage of day 1: Changes in RAC were seen as early as day 5 with RA alone-treated animals having significantly higher counts at days 5, 10, and 15Figure 3
Radial alveolar counts (RAC) as a percentage of day 1:
Changes in RAC were seen as early as day 5 with RA alone-
treated animals having significantly higher counts at days 5,
10, and 15. However, at d30, RA-treated animals had counts
similar to controls. Dex alone-treated animals had signifi-
cantly lower RAC at each time point studied. (*p < 0.05 vs.
saline-treated animals;
†
p < 0.05 vs. CSO-treated animals,
‡
p
= 0.49 vs. saline-treated animals,
§

p = 0.58 vs. CSO-treated
animals) CSO: cottonseed oil.
Respiratory Research 2006, 7:47 />Page 9 of 12
(page number not for citation purposes)
Since the structural effects of Dex administration during
the time of secondary septation are sustained to adult-
hood, the concept of a "critical period" of alveolar devel-
opment was proposed. However, several lines of evidence
support the notion that alveolar growth occurs throughout
life and can be manipulated past the immediate newborn
period. For example, starvation-induced decreases in alve-
olar formation are reversed upon refeeding [
18
]. In addi-
tion, treatment of rats previously exposed to Dex from d3
to d15 with RA from d24 to d36 results in a restitution of
Dex-induced simplification of the distal lung [
11
]. Most
promising for clinical practice, however, is the ability of RA
to stimulate alveolar formation in adult rats after emphy-
sema was induced by intratracheal instillation of elastase
[
19
]. On the other hand, in the present study, the effects of
RA alone were not sustained after discontinuation of treat-
ment.
The mechanisms of RA effects on alveolarization are likely
via changes in the expression of epithelial (e.g. VEGF) and
mesenchymal (e.g. PDGF and TGFβ) growth factors criti-

cal for cell proliferation and differentiation, angiogenesis,
and matrix deposition during lung development [20,21].
In addition, the regulation of free all-trans-RA by RA-bind-
ing proteins and interactions with RA receptors (RAR and
RXR) contribute to appropriate lung development. For
example, RAR-α promotes epithelial cell differentiation
during the progression from pseudoglandular to canalicu-
lar stages of lung development [20]. In addition, RAR-α
also promotes alveolar formation after the perinatal
period [22]. Meanwhile, the expression of RAR-β increases
towards the end of the saccular stage corresponding to an
induction of both type 1 and type 2 epithelial cells [20].
However, RAR-β knockout mice exhibit premature septa-
tion and RAR-β agonist treatment of neonatal rats results
in impaired septation, thereby identifying RAR-β as an
inhibitor of alveolar formation [23]. On the other hand,
impaired distal airspace formation during postnatal lung
development has also been reported in RAR-β knockout
mice [24]. Finally, targeted deletion of RAR-γ in mice is
associated with a decrease in alveolar number, suggesting
the importance of this receptor in the development of
normal alveoli [25]. In our study, while Dex + RA treat-
ment prevented some structural effects seen with Dex
alone, effects due to RA alone were reversible. This sug-
gests that mechanisms are in place to normalize alveolar
structure, but these mechanism(s), in particular those
leading to the reversal of RA effects, are currently
unknown.
To date, there has been a paucity of literature on the effects
of hormone-induced structural changes on pulmonary

function. Srinivasan et al. examined several lung variables
including RR, MV, and TV in sedated animals at 30 to 39
days of life and noted no differences in any treatment
Table 4: Arterial blood gases at d15 and d30: Dex- or RA-treated animals at d15 had a respiratory acidosis with hypercarbia (*p < 0.01
vs. saline/controls) and this was maintained in Dex-treated animals at d30 (*p < 0.01 vs. saline/controls). Day 15 animals given Dex +
RA did not have respiratory acidosis compared to Dex alone pups (
†
p < 0.05 vs. Dex alone). Animals at d30 that had been given Dex +
RA showed a correction of pH and pCO2 (
†
p < 0.05 vs. Dex). Only d15 RA-treated animals had significantly lower pO2 values
compared to controls (**p < 0.05 vs. saline/controls). Values are expressed mean ± SE.
Treatment group pH pCO
2
pO
2
Day 15
Control/Saline (n = 6) 7.39 ± 0.03 41.9 ± 3.2 89.8 ± 6.4
Retinoic acid (RA) (n = 4) 7.29 ± 0.03* 54.2 ± 1.6* 67.3 ± 7.1**
Dexamethasone (Dex) (n = 7) 7.27 ± 0.01* 56.3 ± 3.4* 76.0 ± 9.4
Dex + RA (n = 4) 7.31 ± 0.04
†
50.1 ± 6.0
†
79.3 ± 2.9
Day 30
Control/Saline (n = 12) 7.38 ± 0.01 47.7 ± 1.0 83.5 ± 2.8
Retinoic acid (RA) (n = 10) 7.38 ± 0.01 50.0 ± 1.5 80.1 ± 3.7
Dexamethasone (Dex) (n = 12) 7.33 ± 0.01* 55.5 ± 1.4* 80.9 ± 5.3
Dex + RA (n = 6) 7.38 ± 0.02

†
48.5 ± 2.0
†
83.7 ± 3.9
Table 5: Volumes of displacement on day 15: Lungs were inflated
to 25 cm H
2
O, dissected en bloc and fixed overnight. The
displacement of water by these lungs was determined and
normalized to body weight in grams. Both Dex- and Dex + RA-
treated animals had increased volumes of displacement as
compared to controls and RA-treated animals (* p < 0.01).
Treatment Group – d15 V
disp
/body weight (mL/g)
Control/Saline (n = 9) 43.7 ± 1.28
Retinoic Acid (RA) (n = 7) 46.5 ± 1.28
Dexamethasone (Dex) (n = 6) 53.1 ± 3.13*
Dex + RA (n = 4) 55.5 ± 3.02*
Respiratory Research 2006, 7:47 />Page 10 of 12
(page number not for citation purposes)
group [12]. While our findings of increased lung volumes
in PV curves of Dex-treated animals mirror those of Srini-
vasan et al., our study, performed at day 13 in non-
sedated animals, showed tachypnea and increased TV and
MV in Dex-treated animals. At d15 and d30 in Dex-treated
animals, blood gases obtained in anesthesized, but spon-
taneously breathing animals revealed a respiratory acido-
sis despite an increased MV confirming significant
PV curves at d30: (A1-4.)Figure 4

PV curves at d30: (A1-4.) Best-fit inflation and deflation curves for each treatment group: A significantly increased hysteresis
was noted in the Dex group versus all other groups (p < 0.05). Data points represent individual measurements for each animal.
(B.) Deflation curves for each treatment group generated from an exponential rise model. (C.) A significant increase in maxi-
mum volume (Vmax) existed between Dex vs. Control/Saline curves (*z = 2.05) as well as Dex + RA vs. Control/Saline (*z =
2.05). No difference was found between RA vs. Control/Saline (z = 0.6).
Respiratory Research 2006, 7:47 />Page 11 of 12
(page number not for citation purposes)
compromise in pulmonary function. It is likely that the
respiratory acidosis and consequent tachypnea are due to
increased dead space in affected animals. Interestingly,
while lung volume differences were not resolved, RAC and
blood gases were normalized with Dex + RA treatment,
suggesting that correction of blood gas abnormalities
likely resulted from an increase in the surface area availa-
ble for CO
2
elimination rather than full correction of lung
volumes. Further, the mechanisms leading to the respira-
tory acidosis and lower PaO
2
seen with RA alone treat-
ment at d15 are unknown. However, these changes were
not evident in Dex + RA pups at d15 or at d30 when the
RA alone-induced changes had reversed, suggesting that
the altered architecture seen in RA alone-treated pups may
have contributed to the blood gas abnormalities.
Varying degrees of exercise intolerance have been
described in patients with emphysema [26] and BPD [27].
In our study, no differences in exercise tolerance were
found with any treatment. This suggests that factors other

than altered alveolar structure, such as fibrosis and restric-
tive/obstructive lung disease, may play a significant role in
the diminished exercise tolerance observed in affected
patients.
Lastly, starvation and decreased body weight are associ-
ated with decreased alveolarization [18]. However, in our
studies, while weight was decreased in both Dex- and Dex
+ RA-treated animals, Dex + RA animals had a restitution
of secondary septation, suggesting that distal lung struc-
ture can be manipulated independent of body weight.
This is important in preterm neonates that have signifi-
cantly compromised nutrition and poor weight gain in
addition to an arrest of alveolarization.
Conclusion
In summary, hormonal treatment of rat pups results in
altered lung architecture. This is associated with signifi-
cant structure-function disturbances where Dex-induced
Table 6: Radial alveolar counts (RAC) at d37 and d52: RAC were
significantly lower in Early Dex + Later CSO-treated and higher
in Early Saline + Later RA-treated pups while Early Dex + Later
RA animals were similar to controls. At d52, while RAC
continued to be significantly lower in Early Dex + Later CSO-
treated pups, Early Saline + Later RA effects were lost two weeks
after stopping RA treatment. Values are expressed mean ± SE.
*p < 0.01 vs. d37 Early Saline + Later CSO-treated animals; †p <
0.01 vs. Early Dex + Late CSO; ¶p < 0.01 vs. Early Saline + late
RA. CSO: Cottonseed oil.
Treatment Group Day 37 Day 52
Control (n = 4) 9.1 ± 0.2 9.5 ± 0.2
Early Saline + Late CSO (n = 4) 9.3 ± 0.1 9.4 ± 0.1

Early Saline + Late RA (n = 4) 10.6 ± 0.1* 9.7 ± 0.1¶
Early Dex + Late CSO (n = 4) 7.7 ± 0.2* 7.8 ± 0.1*
Early Dex + Late RA (n = 4) 8.9 ± 0.3† 9.2 ± 0.1†
Effect of delayed RA treatment on alveolarization: Animals were treated with either Dex or saline from d1-14 followed by either CSO or RA from d24-36Figure 5
Effect of delayed RA treatment on alveolarization: Animals were treated with either Dex or saline from d1-14 followed by
either CSO or RA from d24-36. A simplified distal architecture was seen in Dex-treated animals at both d37 (top) and d52
(bottom). RA-treated pups had smaller more numerous alveoli at d37 with changes no longer seen at d52. Early Dex + Later
RA treatment resulted in restored secondary septation that was similar to that of saline controls at d37, and this was sustained
to d52. (all images 100× magnification)
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Respiratory Research 2006, 7:47 />Page 12 of 12
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decreases in alveolarization are associated with increased
lung volumes, CO
2
retention, acidosis, and tachypnea.
Our findings showing that the effects of RA on generation
of alveoli and on gas exchange appear to be time-limited
and reversible may be relevant for the use of RA in treat-

ment of diseases such as BPD or emphysema.
Competing interests
The author(s) declare that they have no competing inter-
ests.
Authors' contributions
SJG was responsible for injecting and harvesting all ani-
mals, measuring radial alveolar counts, performing all
functional and statistical analyses, and drafting the manu-
script. HZ was responsible for generating all the blood gas
and volume of displacement data at day 15, interpretation
and revision of the manuscript. JPF assisted in animal har-
vesting and injections as well as conducting functional
studies. RIG and MHG assisted in obtaining the pressure
volume measurements. AJG assisted in the analysis of PV
curves. RCS conceived the study, participated in its design
and coordination, and helped draft and revise the manu-
script. All authors read and approved the final manu-
script.
Acknowledgements
The authors thank Dr. Phillip L. Ballard for his critical review of the manu-
script. The experiments in this study were supported by NIH grants
HL62858 and HL075930 to RCS. HZ is the recipient of the NIH Pediatric
Scientist Development Award (HD00850).
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